本氏烟NbNAC062的克隆及对马铃薯Y病毒侵染的抑制作用

Cloning of Nicotiana benthamiana NAC062 and Its Inhibitory Effect on Potato Virus Y Infection

【目的】马铃薯Y病毒(potato virus Y,PVY)是危害我国烟草生产的最重要病毒之一,NAC转录因子与植物的抗病、抗逆密切相关,本论文克隆NbNAC062进行生物信息学分析,并研究其在PVY侵染过程中的作用,为烟草抗病毒药剂的开发提供靶标。【方法】以本氏烟(Nicotiana benthamiana)为材料克隆NbNAC062,利用MEGA、UniProt、SMART、TMHMM Server 2.0、Sol Genomics Network、PlantCARE等技术进行生物信息学分析;利用激光共聚焦与实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)明确PVY侵染前后NbNAC062蛋白定位及mRNA表达量变化;基于病毒介导的基因沉默(virus-induced gene silencing,VIGS)和过表达技术,构建pTRV::NbNAC062沉默载体与pEarleyGate100::RFP::NbNAC062过表达载体,采用qRT-PCR和Western blot检测NbNAC062在本氏烟中沉默与过表达后,PVY的积累量变化及未折叠蛋白应答(unfolded protein response,UPR)相关基因BiP的表达差异。【结果】NbNAC062编码646个氨基酸,N端28—179 aa为NAC结构域,129—185 aa为DNA结合区域,C末端621—643 aa为疏水跨膜结构,系统进化树与蛋白序列分析表明本氏烟NbNAC062与渐狭叶烟草NaNAC062亲缘关系最近。NbNAC062启动子中包含脱落酸、茉莉酸甲酯、水杨酸以及逆境响应相关的多种顺式作用元件。PVY侵染激活NbNAC062从细胞膜转移至细胞核,且诱导NbNAC062上调表达。PVY侵染本氏烟5、7 d,处理组NbNAC062 mRNA水平分别为对照组的2.52、1.95倍;PVY侵染3 d,BiP mRNA表达量为对照组的2.39倍,PVY侵染7 d,BiP表达量极显著低于对照组,下调表达56.77%。本氏烟沉默NbNAC062并接种PVY,接种后3、5、7 d,与对照组相比,沉默组PVY CP mRNA上调表达,分别为对照组的2.12、2.41、1.38倍,BiP mRNA表达量则下调,分别下调28.19%、58.11%、10.77%,接种后5、7 d沉默组PVY CP蛋白含量亦显著高于对照组。过表达NbNAC062并接种PVY,接种后24、48、72 h,与对照组相比,过表达组PVY CP mRNA分别下调22.60%、34.51%、36.21%,接种48、72 h,BiP mRNA上调表达,分别为对照组的1.56、1.35倍,过表达组PVY CP蛋白含量亦低于对照组。【结论】NbNAC062属于NAC类膜结合转录因子,可被PVY侵染激活转移至细胞核,可能通过调控UPR相关基因BiP的表达,促进细胞生存,抑制PVY早期侵染。

【Objective】Potato Y virus(PVY)is one of the most important viruses that endanger the tobacco production in China.NAC transcription factors are closely related to plant disease resistance and stress resistance.The objective of this study is to clone NbNAC062,analyze its bioinformatics and research its role in the process of PVY infection,and to provide a target for the development of tobacco antiviral agents.【Method】Nicotiana benthamiana was used as the material to clone NbNAC062,and MEGA,UniProt,SMART,TMHMM Server 2.0,Sol Genomics Network,PlantCARE and other technologies were used for bioinformatics analysis.Laser confocal microscope and quantitative real-time PCR(qRT-PCR)were used to clarify the localization of NbNAC062 protein and the change of NbNAC062 mRNA expression before and after PVY infection.Based on virus-induced gene silencing(VIGS)technology and over-expression technology,the pTRV::NbNAC062 silencing vector and the pEarleyGate100::RFP::NbNAC062 over-expression vector were constructed.qRT-PCR and Western blot were used to detect the changes of PVY accumulation and the expression of unfolded protein response(UPR)related gene BiP after silencing and over-expression in N.benthamiana.【Result】NbNAC062 encodes 646 amino acids,the N-terminal 28-179 aa is the NAC domain,129-185 aa is the DNA binding region,and the C-terminal 621-643 aa is a hydrophobic transmembrane structure.Phylogenetic tree and protein sequence analysis show that N.benthamiana NbNAC062 is closely related to N.attenuata NaNAC062.The NbNAC062 promoter contains a variety of cis-acting elements related to abscisic acid,methyl jasmonate,salicylic acid and stress response.PVY infection activates NbNAC062 to transfer from cell membrane to nucleus and induces NbNAC062 up-regulation of expression.For 5 and 7 days after PVY infection,the NbNAC062 mRNA level in the treatment group was 2.52 and 1.95 times of that of the control group,respectively.For 3 days after PVY infection,the BiP mRNA expression was 2.39 times of that of the control group,and for 7 days after PVY infection,the expression of BiP was significantly lower than that of the control group,which was down-regulated by 56.77%.NbNAC062 was silenced and PVY was inoculated,compared with the control group,the expression of PVY CP mRNA was up-regulated in the silence group at 3,5,and 7 days after inoculation,which was 2.12,2.41,and 1.38 times of that of the control group,respectively.However,the expression of BiP mRNA was down-regulated by 28.19%,58.11%,and 10.77%,respectively.The PVY CP protein content of the silence group was also significantly higher than that of the control group at 5 and 7 days after vaccination.NbNAC062 was over-expressed and PVY was inoculated,compared with the control group,the expression of PVY CP mRNA in the over-expression group at 24,48,72 hours after inoculation was down-regulated by 22.60%,34.51%,and 36.21%,respectively,and BiP mRNA was up-regulated at 48 and 72 hours after inoculation,which was 1.56 and 1.35 times of that of the control group,respectively.The content of PVY CP in the over-expression group was also lower than that of the control group.【Conclusion】NbNAC062 belongs to the NAC class of membrane-bound transcription factors,which can be activated by PVY infection and transferred to the nucleus.It may regulate the expression of the UPR-related gene BiP to promote cell survival and inhibit early PVY infection.